Interactions of inhibition and excitation in the light-evoked currents of X type retinal ganglion cells

The excitatory and inhibitory conductances driving the light-evoked currents (LECs) of cat and ferret ON- and OFF-center X ganglion cells were examined in sliced and isolated retina preparations using center spot stimulation in tetrodotoxin (TTX)-containing Ringer. ON-center X ganglion cells showed an increase in an excitatory conductance reversed positive to +20 mV during the spot stimulus. At spot offset, a transient inhibitory conductance was activated on many cells that reversed near ECl. OFF-center X ganglion cells showed increases in a sustained inhibitory conductance that reversed near ECl during spot stimulation. At spot offset, an excitatory conductance was activated that reversed positive to +20 mV. The light-evoked current kinetics of ON- and OFF-center X cells to spot stimulation did not significantly differ in form from their Y cell counterparts in TTX Ringer. When inhibition was blocked, current-voltage relations of the light-evoked excitatory postsynaptic currents (EPSCs) of both ON- and OFF-X cells were L-shaped and reversed near 0 mV. The EPSCs averaged between 300 and 500 pA at -80 mV. The metabotropic glutamate receptor agonist 2-amino-4-phosphonobutyric acid (APB), was used to block ON-center bipolar cell function. The LECs of ON-X ganglion cells were totally blocked in APB at all holding potentials. APB caused prominent reductions in the dark holding current and synaptic noise of ON-X cells. In contrast, the LECs of OFF-X ganglion cells remained in APB. An increase in the dark holding current was observed. The excitatory amino acid receptor antagonist combination of D-amino-5-phosphono-pentanoic acid (D-AP5) and 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxalinedione (NBQX) was used to block ionotropic glutamate receptor retinal neurotransmission. The LECs of all ON-X ganglion cells were totally blocked, and their holding currents were reduced similar to the actions of APB. For OFF-X ganglion cells, the antagonist combination always blocked the excitatory current at light-OFF; however, in many cells, the inhibitory current at light-ON remained. ON-center X ganglion cells receive active excitation during center illumination, and a transient inhibition at light-OFF. In contrast OFF-center X ganglion cells experience a sustained active inhibition during center illumination, and a shorter increase in excitation at light-offset. Cone bipolar cells provide a resting level of glutamate release on X ganglion cells on which their light-evoked currents are superimposed [corrected].     

Interactions of GYKI 52466 and NBQX with cyclothiazide at AMPA receptors: experiments with outside-out patches and EPSCs in hippocampal neurones

In outside-out patches from cultured hippocampal neurones, glutamate (1 mM) applied for 1 ms evoked currents which rose rapidly (tau(on) 451 +/- 31 micros) to a peak and then deactivated with slower kinetics (1.95 +/- 0.13 ms). Offset time constants were significantly slower with longer application durations (tau(off) 3.10 +/- 0.19, 3.82 +/- 0.25, 4.80 +/- 0.65 and 7.56 +/- 0.65 ms with 10, 20, 100 and 500 ms applications respectively). Desensitization was complete within 100 ms with a similar rate for all application durations (4.74 +/- 0.34 ms with 100 ms applications). GYKI 52466 reduced inward peak currents with an IC50 of 11.7 +/- 0.6 microM and had similar potency on steady-state currents to longer glutamate applications. GYKI 52466 had no significant effect on desensitization or deactivation time constants but caused a modest and significant prolongation of onset kinetics at higher concentrations. Cyclothiazide (100 microM) potentiated steady-state currents 25-fold at 100 ms and caused a modest but significant slowing in onset kinetics (601 +/- 49 micros with 1 ms applications) but a more pronounced prolongation of deactivation time constants (5.55 +/- 0.66 ms with 1 ms applications). In 50% of neuronal patches cyclothiazide completely eliminated desensitization. In those patches with residual desensitization, the rate was not significantly different to control (5.36 +/- 0.43 ms with 100 ms applications). Following 100 ms applications of glutamate, GYKI 52466 had IC50s of 11.7 +/- 1.1 microM and 75.1 +/- 7.0 microM in the absence and presence of cyclothiazide (100 microM) respectively. Onset kinetics were slowed from 400 +/- 20 micros to 490 +/- 30 micros by cyclothiazide (100 microM) and then further prolonged by GYKI 52466 (100 microM) to a double exponential function (tau(on1) 1.12 +/- 0.13 ms and tau(on2) 171.5 +/- 36.5 ms). GYKI 52466 did not re-introduce desensitization but concentration-dependently weakened cyclothiazide's prolongation of deactivation time constants (1 ms applications: 5.01 +/- 0.71, 4.47 +/- 0.80 and 2.28 +/- 0.64 ms with GYKI 52466 30, 100 and 300 microM respectively). NBQX reduced peak current responses with an IC50 of 28.2 +/- 1.3 nM. Paradoxically, steady-state currents with 500 ms applications of glutamate were potentiated from 3.3 +/- 1.2 pA to 29.4 +/- 6.4 pA by NBQX (1 nM). Higher concentrations of NBQX then antagonized this potentiated response. The potency of NBQX in antagonizing steady-state currents to 500 ms applications of glutamate (IC50 120.9 +/- 30.2 nM) was 2-fold less than following 100 ms applications (IC50 67.7 +/- 2.6 nM). NBQX had no effect on rapid onset, desensitization or deactivation time constants. However, a slow relaxation of inhibition was seen with longer applications. NBQX was 2-5-fold less potent against inward currents in the presence of cyclothiazide (100 microM) depending on the application duration but had no effect on the rapid onset, desensitization or deactivation time constants. The same relaxation of inhibition was seen as with NBQX alone. NBQX (1 microM) reduced AMPA receptor-mediated EPSC amplitude to 7 +/- 1% of control with no effect on kinetics. Cyclothiazide (330 microM) caused a 2.8-fold prolongation of the decay time constant (control 26.6 +/- 2.2 ms, cyclothiazide 74.2 +/- 7.6 ms, n = 9). Additional application of NBQX (1 microM) partly reversed this prolongation to 1.9 fold (47.7 +/- 2.5 ms, n = 5). These results support previous findings that cyclothiazide also allosterically influences AMPA receptor agonist/antagonist recognition sites. There were no interactions between NBQX and cyclothiazide on desensitization or deactivation time constants of glutamate-induced currents but clear interactions on EPSC deactivation kinetics. This raises the possibility that the interactions of NBQX, GYKI 52466 and cyclothiazide on AMPA-receptor-mediated EPSC kinetics observed are due to modulation of glutamate-release at presynaptic AMPA receptors.     

The mechanism by which NBQX enhances NMDA currents in retinal ganglion cells

When the quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline), a KA/AMPA antagonist, is bath applied to the tiger salamander retina, a paradoxical action is evident in the light-evoked synaptic responses of ganglion cells: NBQX enhances excitatory synaptic currents at light onset observed under whole-cell voltage-clamp conditions in a perfused retinal slice preparation. This observation was surprising because synaptic inputs into ganglion cells that are mediated by KA/AMPA receptors are entirely blocked by NBQX. Thus, the NBQX-enhanced current is entirely mediated by NMDA receptors. The purpose of this study was to determine the mechanism(s) by which blocking KA/AMPA receptors appears to enhance NMDA currents. Using hyperosmotic sucrose stimulation to activate neurotransmitter release from the inner retina, we observed that NBQX augmented the sucrose-evoked response, suggesting that at least a component of this enhancement may reside in the inner retina. NBQX does not enhance NMDA currents activated by bath applied NMDA, demonstrating that the NBQX-induced enhancement does not result from modulation of NMDA receptors. Voltage-clamp studies, carried out at the appropriate holding potential, indicate that NBQX enhances glutamatergic transmission and reduces inhibitory inputs onto ganglion cells. In the presence of strychnine and picrotoxin, the NBQX-induced enhancement of NMDA currents is eliminated, suggesting that NBQX facilitates the expression of NMDA currents by a selective and partial reduction of inhibitory mechanisms. Additional studies suggest that part of the NMDA enhancement by NBQX is evident at the postsynaptic level, but a presynaptic component probably also participates, perhaps at the level of bipolar cell terminals. One way to account for this observation is to assume that a subpopulation of inhibitory amacrine cells requires KA/AMPA receptors exclusively for their synaptic activation: previous studies of sustained amacrine cells support this interpretation. Thus the NBQX-induced enhancement phenomenon may reflect a network-selective distribution of NMDA and KA/AMPA receptors among third-order neurons.     

Orthodontic treatment planning for inclusion of the third molar in the dental arches: Part I

The properties of nicotinic acetylcholine receptors (AChRs) on cultured rat superior cervical ganglion (SCG) neurons were analysed. AChR agonists [1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), cytisine] were applied to whole cells within 70ms. The desensitization rate of whole-cell currents during constant application of DMPP varied between neurons. The time course of desensitization was fitted by double exponentials with time constants kfast, of between 0.35 and 0.55s, and kslow, of 3-5s. By exchanging intracellular chloride for caesium methanesulphonate, the possibility of interference by a calcium-activated chloride current was excluded. In cells that exhibited a slowly desensitizing current during the application 20 microM DMPP, equimolar cytisine induced a larger peak current compared to the response to DMPP, while in cells with rapidly desensitizing DMPP-induced currents the response to equimolar cytisine was smaller. The differences in desensitization rates and agonist potencies are due to different functional properties of AChR subtypes, as indicated by currents recorded from outside-out patches upon rapid agonist application and removal (2ms each). The results indicate the presence of two distinct AChR subtypes on SCG neurons: one with a fast and one with a slow activation/desensitization rate, but both with similar single-channel conductances. Slow activation/desensitization was found to be associated with a high potency of cytisine/low potency of DMPP. For AChRs with rapid activation/desensitization kinetics the agonist potencies were reversed.     

Cable properties of cultured hippocampal neurons determined from sucrose-evoked miniature EPSCs

1. The passive cable properties of rat hippocampal neurons in dissociated culture were studied using focal application of hypertonic solution to locally elicit miniature excitatory postsynaptic currents (mEPSCs) on the soma and dendrites. Neurons were filled with Lucifer yellow and portions of their dendritic trees were measured. 2. The average mEPSC measured at the soma appeared smaller and slower as the site of sucrose application was made more distal. Normalizing to a 1-micron diam dendrite, the mean mEPSC peak amplitude and charge was reduced e-fold in 170 and 1,000 microns, respectively, and the mean mEPSC decay time constant was increased e-fold in 150 microns. However, for any particular sucrose site, individual mEPSCs varied widely in their amplitudes and time courses. Plots of individual peak amplitudes versus half-width or rise time showed much overlap for mEPSCs originating from sites as much as 100 microns apart. This suggests that use of such plots to estimate the electrotonic location of synaptic currents is highly prone to error. 3. Averaged mEPSCs recorded when applying sucrose at the soma were poorly fitted by an alpha function but were well-described by an equation of the form mxh, where m incorporates a rise-time constant tau 1 and h a decay time constant tau 2. Averaged fits to mean mEPSCs elicited at the somas of five cells gave (mean +/- SE): peak conductance = 832 +/- 126 pS, tau 1 = 0.29 +/- 0.06 ms, tau 2 = 3.03 +/- 0.24 ms, x = 4.7 +/- 0.7. 4. For three cells, the entire dendritic branch to which sucrose was applied was measured and used to construct a passive cable model. The specific membrane resistance (Rm) and intracellular resistivity (Ri) were varied systematically in the model (assuming membrane capacitance Cm = 1 microF/cm2) to search for the best agreement between the mean mEPSCs and the model. Optimal Rm was found to lie in the range 20-30 k omega cm2, Ri in the range 100-200 omega cm. 5. These results confirm those obtained by other methods and emphasize the considerable cable filtering of fast electrical events in cultured hippocampal neurons.     

AII amacrine cell population in the rabbit retina: identification by parvalbumin immunoreactivity

Parvalbumin (PV) is a calcium-binding protein localized to selected neurons in the nervous system, including the retina. This investigation evaluated the distribution of PV immunoreactivity in the rabbit retina using immunohistochemistry with a monoclonal antibody directed to carp PV. In the inner nuclear layer (INL), PV immunoreactivity was present in horizontal and amacrine cells. In the ganglion cell layer, PV immunostaining was confined to somata that are likely to be both displaced amacrine cells and ganglion cells. PV-immunoreactive (IR) amacrine cells were positioned in the proximal INL adjacent to the inner plexiform layer (IPL). These cells usually gave rise to a single primary process, which arborized into two distinct bands in the IPL. In sublamina a, the processes were thin and had large, irregular endings. In sublamina b, multiple processes branched from the primary process and were characterized by varicosities and spines. PV-IR amacrine cell bodies measured from 8 to 10 microns in diameter. Their density was highest in the visual streak and lowest in the periphery of the superior retina. The average number of PV-IR amacrine cells was 464,045 cells per retina (N = 3), and the average regularity index of the PV-IR cell mosaic was 3.23. PV-IR amacrine cells were further characterized by double-label immunofluorescence experiments using antibodies to PV and tyrosine hydroxylase (TH). Varicose TH-IR processes were in close apposition to many PV-IR amacrine cells and often formed "ring structures" around them. Together, these morphological, quantitative, and histochemical observations indicate that PV immunoreactivity in the INL is localized predominantly to AII amacrine cells, and therefore it is a valuable marker for the identification of this cell type.     

Desensitizing glutamate receptors shape excitatory synaptic inputs to tiger salamander retinal ganglion cells

AMPA/kainate (KA) receptors mediate a component of ganglion cell excitatory postsynaptic currents (EPSCs). We investigated whether desensitization at these receptors contribute to the shape of transient EPSCs in ON-OFF ganglion cells. Whole-cell, voltage-clamp recordings were made from ganglion cells in the retinal slice or in isolation. EPSCs were evoked by either stimulating the slice with light or puffing K+ at the outer plexiform layer (OPL). The AMPA/KA receptor-mediated component of the EPSCs was isolated by including NMDA receptor antagonists in the bath. Strychnine and picrotoxin blocked inhibitory inputs. In isolated ganglion cells, cyclothiazide (10 microM), which blocks desensitization in non-NMDA receptors, enhanced both the amplitude and the duration of currents evoked by puffs of AMPA or glutamate. EPSCs evoked by K(+)-puffs in the OPL were also enhanced by cyclothiazide (30 microM). When AMPA/KA receptors were blocked with NBQX (10 microM), no enhancement of the EPSCs by cyclothiazide was observed, indicating that cyclothiazide did not act presynaptically. Cyclothiazide also enhanced the amplitude and duration of both the ON and OFF light-evoked (L-) EPSCs recorded in ON-OFF ganglion cells. Current-voltage relationships showed the enhancement was not voltage dependent. When control and enhanced responses where normalized, it was observed that the rate of desensitization of both the ON and OFF L-EPSCs was decreased by cyclothiazide. Cyclothiazide selectively enhanced the AMPA/KA receptor-mediated component of ganglion cells EPSCs, suggesting that desensitization of AMPA/KA receptors shape transient L-EPSCs.     

Desensitizing GABAC receptors on carp retinal bipolar cells

Bicuculline- and baclofen-insensitive GABA receptors (GABAC receptors) on bipolar cells acutely dissociated from carp retina were investigated with using the whole-cell patch-clamp recording technique. The currents of these cells mediated by GABAC receptors showed striking desensitization, even at low concentrations of GABA. Both the time constant tau of the GABAC current decay and the extent of desensitization were significantly different from that of GABAC receptors previously observed in other retinas and elsewhere in the CNS, suggesting that the GABAC receptors of carp bipolar cells might be distinct in intracellular mechanisms and subunit composition.     

OFF-alpha and OFF-beta ganglion cells in cat retina: II. Neural circuitry as revealed by electron microscopy of HRP stains

An OFF-center alpha and an OFF-center beta ganglion cell in cat retina, which had been recorded from and intracellularly stained with horseradish peroxidase (HRP) were examined by serial section electron microscopy. We counted synapses and identified presynaptic neurons to the HRP-stained cells in 20 microns radial slices through the centers of their dendritic trees. Presynaptic amacrine and bipolar cells were identified on cytological criteria known from previous studies. The OFF-beta cell with a 62 microns dendritic arbor, restricted to S1 and S2 (sublamina a) of the inner plexiform layer (IPL), received 38% bipolar and 62% amacrine cell synapses. The bipolar input was from both cb1 and cb2 cone bipolar types. Input from three distinct amacrine cell types occurred upon the dendrites, namely from: (1) AII amacrine lobular appendages, (2) large pale amacrine profiles (possibly A2 or A3 cells), and (3) small, dark amacrine types (possibly A8 cells). Large pale amacrine profiles (possibly A13) were found on the cell body and apical dendrite in sublamina b of the IPL. In addition, several amacrine profiles synapsed directly on the sides and base of the cell body in the ganglion cell layer. We estimate that the complete dendritic tree of this beta cell received about 1,000 synapses contributed by 12-14 bipolar cells, 7-10 AII amacrines and 28-41 other amacrine cells. The OFF-alpha cell had a dendritic tree size of 680 x 920 microns. A 250 microns length of two major dendrites stratifying narrowly in S2 of the IPL was reconstructed. Amacrine cells provided most of the synaptic input (80%). This input came from: (1) AII amacrine lobular appendages, (2) amacrines exhibiting large, pale synaptic profiles (possibly A2 or A3 cells), (3) pale amacrines with large mitochondria and a few neurotubules (unknown type), and (4) densely neurotubule-filled amacrine profiles (possibly A19 cells). A large pale amacrine cell type (possibly A13) provided synaptic input to the cell body as a serial synaptic intermediary with rod bipolar cells. Cone bipolar synapses were from only one type of cone bipolar, the cb2 type and formed 20% of the total synaptic input. We estimate that a minimum of 142 bipolar cells, 256 AII amacrine cells and 1,011 other amacrine cells, altogether providing 6,000-10,000 synapses, converged on the dendritic tree of this OFF-alpha cell.     

The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

Membrane currents evoked by ionotropic glutamate receptor agonists in rod bipolar cells in the rat retinal slice preparation

1. With the use of the whole cell voltage-clamp technique, I have recorded the current responses to ionotropic glutamate receptor agonists of rod bipolar cells in vertical slices of rat retina. Rod bipolar cells constitute a single population of cells and were visualized by infrared differential interference contrast video microscopy. They were targeted by the position of their cell bodies in the inner nuclear layer and, after recording, were visualized in their entirety by labeling with the fluorescent dye Lucifer yellow, which was included in the recording pipette. To study current-voltage relationships of evoked currents, voltage-gated potassium currents were blocked by including Cs+ and tetraethylammonium+ in the recording pipette. 2. Pressure application of the non-N-methyl-D-aspartate (non-NMDA) receptor agonists kainate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) from puffer pipettes evoked a long-latency conductance increase selective for chloride ions. When the intracellular chloride concentration was increased, the reversal potential changed, corresponding to the change in equilibrium potential for chloride. The response was evoked in the presence of 5 mM Co2+ and nominally O mM Ca2+ in the extracellular solution, presumably blocking all external Ca2(+)-dependent release of neurotransmitter. 3. The long latency of kainate-evoked currents in bipolar cells contrasted with the short-latency currents evoked by gamma-aminobutyric acid (GABA) and glycine in rod bipolar cells and by kainate in amacrine cells. 4. Application of NMDA evoked no response in rod bipolar cells. 5. Coapplication of AMPA with cyclothiazide, a blocker of agonist-evoked desensitization of AMPA receptors, enhanced the conductance increase compared with application of AMPA alone. Coapplication of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the response to kainate and AMPA, indicating that the response was mediated by conventional ionotropic glutamate receptors. 6. The conductance increase evoked by non-NMDA receptor agonists could not be blocked by a combination of 100 microM picrotoxin and 10 microM strychnine. Application of the GABAC receptor antagonist 3-aminopropyl (methyl)phosphinic acid (3-APMPA) strongly reduced the response, and coapplication of 500 microM 3-APMPA and 100 microM picrotoxin completely blocked the response. These results suggested that the conductance increase evoked by non-NMDA receptor agonists was mediated by release of GABA and activation of GABAC receptors, and most likely also GABAA receptors, on rod bipolar cells. 7. Kainate responses like those described above could not be evoked in bipolar cells in which the axon had been cut somewhere along its passage to the inner plexiform layer during the slicing procedure. This suggests that the response was dependent on the integrity of the axon terminal in the inner plexiform layer, known to receive GABAergic synaptic input from amacrine cells. 8. The results indicate that ionotropic glutamate receptors are not involved in mediating synaptic input from photoreceptors to rod bipolar cells and that an unconventional mechanism of GABA release from amacrine cells might operate in the inner plexiform layer.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.     

ON-OFF amacrine cells in cat retina

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b) but extends some dendrites to sublamina a. Both of these cell types have axon-like processes that extend > 800 microns from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 +/- 0.3 mm diameter) that were broader than their dendritic arbors (425 +/- 35 microns diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-alpha cell.     

High levels of extracellular glutamate are present in retina during neonatal development

The three major classes of neurons which comprise the primary visual pathway in retina are glutamatergic. These cells are generated in two separate developmental stages, with one subclass of photoreceptors (cones) and ganglion cells generated before birth; and the other subclass of photoreceptors (rods) and bipolar cells generated during the first week after birth. Gas chromatography/mass spectroscopy analysis coupled with a new method for collecting small samples of extracellular fluids from retina were used to determine the levels of endogenous glutamate present during differentiation and synaptogenesis of these different cell types. As expected the total retinal content of glutamate increased during the postnatal period in synchrony with the generation and maturation of glutamatergic cells. However, a significant proportion of the endogenous pool was found extracellularly at birth. Intracellular glutamate is localized within cell bodies and growing processes of cones and ganglion cells at this time but few glutamatergic synapses are present. The extracellular concentration of glutamate actually declined during the most active period of synaptogenesis, reaching very low levels in the adult. The high concentrations of extracellular glutamate in neonatal retina could play an important role in a variety of developmental events such as dendritic pruning, programmed cell death and neurite sprouting.     

Localization and function of five glutamate transporters cloned from the salamander retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.     

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Two different types of action potentials were observed among the pyramidal cells and interneurons in cat motor cortex: the narrow action potentials and the wide action potentials. These two types of action potentials had similar rising phases (528.8 +/- 77.0 vs 553.1 +/- 71.8 mV/ms for the maximal rising rate), but differed in spike duration (0.44 +/- 0.09 vs 1.40 +/- 0.39 ms) and amplitude (57.31 +/- 8.22 vs 72.52 +/- 8.31 mV), implying that the ionic currents contributing to repolarization of these action potentials are different. Here we address this issue by pharmacological manipulation and using voltage-clamp technique in slices of cat motor cortex. Raising extracellular K+ concentration (from 3 mM to 10 mM), applying a low dose of 4-aminopyridine (2-200 microM) or administering a low concentration of tetraethylammonium (0.2-1.0 mM) each not only broadened the narrow action potentials, but also increased their amplitudes. In contrast, high K+ medium or low dose of tetraethylammonium only broadened the wide action potentials, leaving their amplitudes unaffected, and 4-aminopyridine had only a slight broadening effect on the wide spikes. These results implied that K+ currents were involved in the repolarization of both types of action potentials, and that the K+ currents in the narrow action potentials seemed to activate much earlier than those in the wide spikes. This early activated K+ current may counteract the rapid sodium current, yielding the extremely brief duration and small amplitude of the narrow spikes. The sensitivity of the narrow spikes to 4-aminopyridine may not be mainly attributed to blockade of the classical A current (IA), because depolarizing the membrane potential to inactivate IA did not reproduce the effects of 4-aminopyridine. Blockade of Ca2+ influx slowed the last two-thirds repolarization of the wide action potentials. On the contrary, the narrow action potentials were not affected by Ca(2+)-current blockers, but if they were first broadened by 4-aminopyridine or tetraethylammonium, subsequent application of Ca(2+)-free medium caused further broadening, suggesting that the narrow action potentials were too brief to activate the Ca(2+)-activated potassium currents for their repolarization. Therefore, the effects of low concentrations of tetraethylammonium on the narrow spikes appeared to be mainly due to blockade of an outward current that was different from the tetraethylammonium-sensitive Ca(2+)-activated potassium current (IC). In the neurons with the narrow spikes, voltage-clamp experiments revealed two voltage-gated outward currents that were sensitive to tetraethylammonium and 4-aminopyridine, respectively. Both currents were activated rapidly following the onset of depolarizing steps. Interestingly, the tetraethylammonium-sensitive current was a transient outward current that inactivated rapidly (tau < or = 5 ms), while the 4-aminopyridine-sensitive current was relatively persistent during maintained depolarization. The 4-aminopyridine-sensitive current did not show obvious inactivation even at membrane potential of -40 mV, which completely inactivated the transient tetraethylammonium-sensitive, current. The results indicate that different potassium currents are involved in the repolarization of the narrow and wide action potentials in cat motor cortex. A novel tetraethylammonium-sensitive transient outward current and a 4-aminopyridine-sensitive outward current are responsible for the short duration and small amplitude of the narrow action potentials in the interneurons and some of the layer V pyramidal cells. These two currents are voltage-gated and Ca(2+)-independent. For the wide action potentials that characterize most pyramidal neurons, a Ca(2+)-independent tetraethylammonium-sensitive outward current and a Ca(2+)-activated potassium current are the main contributors to their repolarization.     

Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons

Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau >/= 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time approximately 0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.     

Two distinct nicotinic receptors, one pharmacologically similar to the vertebrate alpha7-containing receptor, mediate Cl currents in aplysia neurons

Ionotropic, nicotinic receptors have previously been shown to mediate both inhibitory (Cl-dependent) and excitatory (cationic) cholinergic responses in Aplysia neurons. We have used fast perfusion methods of agonist and antagonist application to reevaluate the effects on these receptors of a wide variety of cholinergic compounds, including a number of recently isolated and/or synthesized alpha toxins [alpha-conotoxin (alphaCTx)] from Conus snails. These toxins have been shown in previous studies to discriminate between the many types of nicotinic receptors now known to be expressed in vertebrate muscle, neuroendocrine, and neuronal cells. One of these toxins (alphaCTx ImI from the worm-eating snail Conus imperialis) revealed that two kinetically and pharmacologically distinct elements underlie the ACh-induced Cl-dependent response in Aplysia neurons: one element is a rapidly desensitizing current that is blocked by the toxin; the other is a slowly desensitizing current that is unaffected by the toxin. The two kinetically defined elements were also found to be differentially sensitive to different agonists. Finally, the proportion of the rapidly desensitizing element to the sustained element was found to be cell-specific. These observations led to the conclusion that two distinct nicotinic receptors mediate Cl currents in Aplysia neurons. The receptor mediating the rapidly desensitizing Cl-dependent response shows a strong pharmacological resemblance to the vertebrate alpha-bungarotoxin-sensitive, alpha7-containing receptor, which is permeable to calcium and mediates a rapidly desensitizing excitatory response.     

Ability of retinal Müller glial cells to protect neurons against excitotoxicity in vitro depends upon maturation and neuron-glial interactions

Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell-specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co-cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron-glial signalling through glutamate-elicited responses.